Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
RESEARCH
Open Access
Biotransformation of ginsenoside Rb1 via the gypenoside pathway by human gut bacteria Hong Shen1,2†, Weng-Im Leung1†, Jian-Qing Ruan1, Song-Lin Li2, Jacky Pui-Cheong Lei3, Yi-Tao Wang1 and Ru Yan1*
Abstract Background: Bacterial conversion of ginsenosides is crucial for the health-promoting effects of ginsenosides. Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria in vitro. Methods: The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis. Results: When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process. Conclusions: Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria in vitro.
Background Ginsenosides are the major bioactive components in Panax, including Panax ginseng, Panax quinquefolium, and Panax notoginseng [1], which are the most popular herbs used for medicinal and nutritional purposes in China, Japan, Korea, and some Western countries. Two dammarane-type triterpene O-glycosides, namely 20(S)protopanaxadiol (PPD) and 20(S)-protopanaxatriol, are the major ginsenosides in different parts of Panax, including the flower bud, berry, crown, rootlet, side root, seed bud, seed, stem, and leaf [2,3], and their differently processed herbal products [4]. As one of the most important PPDtype ginsenosides, ginsenoside Rb1 (Rb1) has exhibited * Correspondence: [email protected] † Equal contributors 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China Full list of author information is available at the end of the article
various pharmacological activities, including neuroprotective, antitumor, cardiovascular-protective, and anti-aging effects, in many in vitro and in vivo models [5-7]. The intestinal absorption of glycosides in plants or foods is poor, while their metabolites generated by intestinal microflora are more permeable and bioactive [8-11]. The oral bioavailability of PPD-type ginsenosides is generally low (98% (HPLCUV). The BBL™ Brain Heart Infusion (BHI) medium,
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GasPak™ EZ anaerobe container system with an indicator, and GasPak™ EZ large incubation container were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). L-cystine was purchased from Research Organics Inc. (Cleveland, OH, USA). Dimethyl sulfoxide (DMSO), bovine hemin, and vitamin K1 were supplied by SigmaAldrich (St. Louis, MO, USA). Methanol, 1-butanol, and acetonitrile were HPLC-grade and purchased from Merck (Darmstadt, Germany). Dulbecco’s phosphate-buffered saline (PBS) was provided by Life Technologies (Carlsbad, CA, USA). Hydrochloric acid and sodium carbonate (analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared in-house using a Milli-Q purification system (Millipore, Bedford, MA, USA). Instrumentation and analytical conditions
Chromatographic separation was performed on an Agilent 1200 Series HPLC apparatus (Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, a binary pump, and an autosampler, and connected to a diode array detector (DAD). Samples were loaded onto an Alltima C18 column (250 × 4.6 mm, 5 μm). The column temperature was maintained at 25°C. The mobile phase consisted of water (A) and acetonitrile (B) and flowed at 1.5 mL/min. The following gradient elution program was used for samples obtained from bacterial incubations: 010 min, 19-20% B; 10-28 min, 20-30% B; 28-40 min, 3060% B; 40-48 min, 60-100% B. The elution program was modified when the incubations of ginsenosides with SGF were analyzed as follows: 0-10 min, 19-20% B; 10-28 min, 20-30% B; 28-35 min, 30-60% B; 35-45 min, 60-100% B. Rb1 and its bacterial metabolites were monitored at 203.4 nm and their UV absorbance was recorded over 200-400 nm. The injection volume was 5 μL. MS analysis was performed on an liquid chromatograph/mass selective detector (LC/MSD) Trap system (Agilent Technologies, Palo Alto, CA, USA) equipped with an ion-trap mass spectrometer with an electrospray ionization (ESI) interface. Agilent ChemStation for LC 3D System software (Rev. B. 01. 03-SR2) (Agilent Technologies, Santa Clara, CA, USA) was used for instrument control, data acquisition, and data management. Except for replacement of the water with 50 mM ammonium acetate in water, the mobile phase composition and gradient elution program were the same as those for the above HPLC analysis method for human gut bacteria samples. The mass spectrometer was operated in the negative ion mode with the following conditions: drying gas (N2), 8 L/min; temperature, 325°C; nebulizer pressure, 30 psi; scan range, m/z 100-1400. The ESIMS/MS conditions were as follows: negative ion mode; separation width, 4; fragment amplification, 1.0 V; scan range, m/z 100-1400.
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
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Preparation of human gut bacteria
Inter-individual variations in the gypenoside and Rd pathways were investigated by incubating Rb1 with gut bacteria suspensions from different individuals under the same conditions described above, and the incubated samples collected at 18 h were processed as described above. Samples were analyzed by HPLC-UV, and the peak area ratio data of each analyte were collected for comparison.
The medium was prepared according to our previous report [33]. Briefly, 100 mL of autoclaved BHI medium (3.7 g/100 mL) was supplemented with 0.05 mg of vitamin K1, 0.5 mg of bovine hemin, and 50 mg of L-cystine. The fecal samples (healthy Chinese, 18-92 years old) were provided by Kiang Wu Hospital and the experimental protocol is approved by Kiang Wu Hospital and University of Macau before conduct. Human gut bacteria suspensions were prepared at 4°C according to our previous report [33] with minor modifications. Fecal samples were freshly collected and pooled at 1 g each for identification of Rb1 metabolic pathways, or individually processed at 5 g each for characterization of individual variations in Rb1 metabolism. The individual or pooled fecal samples were mixed with 20 mL of BHI medium and the resulting suspensions were centrifuged at 200 × g for 5 min. The supernatants were then centrifuged at 5000 × g for 30 min. The human gut bacteria suspensions were obtained by resuspension of the bacterial precipitates in 5 mL of BHI medium. Biotransformation of Rb1 by human gut bacteria
Biotransformation assays of Rb1 were conducted with pooled or individual human gut bacteria samples to examine the existence of the gypenoside pathway and compare it with the Rd pathway. A high concentration of Rb1 was used to capture the minor formation and quantitative variations in the metabolites formed from the minor gypenoside pathway based on the great individual variations in gut bacteria compositions and metabolic capabilities [34]. The anaerobic incubation system contained 250 μL of gut bacteria suspension, 100 μL of Rb1 in DMSO (final concentration, 100 mM Rb1), and 4.75 mL of BHI medium. The incubation system was maintained in an anaerobic state at 37°C using the GasPak™ EZ anaerobe container system. The metabolic pathway and time-course of Rb1 metabolism by human gut bacteria were characterized in five independent experiments using pooled samples. In each experiment, the reaction of Rb1 with a human gut bacteria suspension was conducted in duplicate. Control reactions were conducted in parallel by adding 250 μL of PBS (pH 7.0) instead of gut bacteria suspension. The reactions were terminated at 0, 2, 4, 12, 18, 24, 36, and 48 h, respectively, by adding 15 mL of 1-butanol followed by immediate centrifugation at 5000 × g for 30 min to remove the bacteria. The resulting supernatants were mixed with 100 μL of ginsenoside Rg1 (final concentration, 100 mM) as an internal standard (IS), and each mixture was extracted twice with water-saturated 1-butanol. The organic layers were combined and dried by rotary evaporation (BUCHI, Flavil, Switzerland). The residue was reconstituted in 0.2 mL of methanol, and an aliquot (5 μL) was subjected to HPLC-UV or HPLC-MS analysis.
Biotransformation of Rd and G-XVII by human gut bacteria
The metabolism of the two positional isomers Rd and G-XVII by human gut bacteria was investigated by incubation with the same human gut bacteria sample in parallel to identify the pathways between Rd/G-XVII and Cpd K. The anaerobic incubation system contained 20 μL of gut bacteria suspension, 10 μL of ginsenoside Rd or G-XVII (final concentration, 100 μM Rd or GXVII), and 170 μL of BHI medium. The concentrations of Rd and G-XVII were determined based on the high amounts of all metabolites determined from a preliminary study. Each reaction was conducted in triplicate. Control reactions without gut bacteria were conducted in parallel. Reactions were terminated at 0, 2, 4, 6, 8, 12, 18, 24, 36, and 48 h, respectively, by adding 1.2 mL of 1-butanol followed by centrifugation at 5000 × g for 30 min to remove the bacteria. After adding 10 μL of ginsenoside Rg1 (final concentration, 100 μM Rg1) as an internal standard, the resulting mixture was processed in the same manner described above before being subjected to HPLC-UV analysis. Stabilities of Rb1 and Rd in SGF
SGF was prepared by diluting 3.84 mL of hydrochloric acid to 1000 mL with deionized water (pH 1.2) according to the Chinese Pharmacopoeia [35]. Ten microliters of ginsenoside Rb1 or Rd in DMSO (final concentration, 71.4 μM) was mixed with 200 μL of SGF, and kept at 37°C for different time intervals (0, 20, 40, 60, 90, 150, 180 and 300 min) before adding 70 μL of 0.1 mol/L Na2CO3 to examine the acidic stability. Each reaction was conducted in triplicate. The mixture was shaken for 1 min by vortexing and centrifuged at 14,000 × g for 5 min. The upper layer was decanted and an aliquot (20 μL) was subjected to HPLC-UV analysis. Calibration curves of Rb1 and its metabolites Rd, F2, G-XVII, and Cpd K in human gut bacteria
Serial concentrations of Rb1, Rd, G-XVII, F2, and Cpd K were prepared in DMSO, and mixed with a pooled human gut bacteria suspension and BHI medium as described in the section entitled Biotransformation of Rb1 by Human Gut Bacteria. The resulting mixtures were
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
immediately extracted with 1-butanol and centrifuged (5000 × g, 30 min). The organic layers were processed in the same manner described above and then subjected to HPLC-UV analysis. Calibration curves were constructed by plotting the peak area ratio of each analyte to the IS (Y) as a function of the analyte concentration (X) in the reaction system. For measurements of the intra-day precision and accuracy, three concentration levels for each standard (low, medium, and high) were prepared as quality control (QC) samples and analyzed on the same day. The inter-day
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precision and accuracy were determined with the same three QC samples on three consecutive days. Calibration curves of ginsenosides in SGF
A mixed stock solution of Rb1, Rd, G-XVII, F2, Rg3, Cpd K, and Rh2 was prepared in DMSO. A series of working solutions were prepared with DMSO using dilution factors of 2.5, 5, 10, 20, 40, and 80 from the stock solution. An aliquot (10 μL) of each working solution was spiked with 200 μL of SGF and processed with 70 μL of 0.1 mol/L Na2CO3 as described above. The
Figure 1 HPLC-UV chromatograms of (A) mixed ginsenoside standards, and incubated samples of Rb1 with (B) pooled gut bacteria and intestinal bacteria from (C) a 65-year-old male, (D) a 44-year-old male, and (E) a 54-year-old female. M1, ginsenoside Rd; M2, gypenoside XVII; M3, ginsenoside F2; M4, gypenoside LXXV; M5, compound K.
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
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sample (20 μL) was injected into the HPLC instrument. A calibration curve was obtained by plotting the peak area of each analyte (Y) against its concentration in SGF (X). Data analysis
Data were presented as the means ± standard deviation (SD) of triplicate analyses (experiments with pooled human gut bacteria or SGF) or individual measurements (experiments on inter-individual variations). Sex and age differences in ginsenoside Rb1 metabolism were compared by Student’s t-test. Values of P < 0.05 were considered statistically significant.
Results Metabolism of ginsenoside Rb1 by pooled human gut bacteria
Fecal samples from 50 healthy volunteers (25 males and 25 females, range, 45.7-73.7 years) were collected and pooled at 10 individuals each to prepare five human gut bacteria pools. When Rb1 was incubated with pooled human gut bacteria, five peaks (M1-M5) were observed at 35.1, 35.6, 38.2, 39.7, and 44.4 min, respectively, which were absent in control reactions (Figure 1). Under the developed analytical conditions, Rb1 and its metabolites were well-separated from the endogenous interferences of the in vitro anaerobic incubation system. The retention times and characteristic CID fragment ions of the ginsenoside standards and Rb1 metabolites are summarized in Table 1. Rb1 was eluted at 33.2 min and showed its pseudomolecular ion at m/z 1107 ([M-H]-) and chloride adduct ion at m/z 1143 ([M + Cl]-), corresponding to a molecular weight of 1108 Daltons. The fragment ions at m/z 945 ([M-H2O-H]-), m/z 783 ([M-2H2O-H]-), and m/z 621 ([M-3H2O-H]-) in the CID spectrum corresponded to loss of one, two, and three glucose molecules from Rb1, respectively.
The retention times and mass spectral profiles of M1 (35.1 min), M3 (38.2 min), and M5 (44.4 min) were identical to those of Rd, F2, and Cpd K, respectively. The retention time and mass spectral profile of M2 (35.6 min) were identical to those of G-XVII, which was formed from Rb1 by hydrolytic removal of one glucose molecule from the C-3 position of Rb1. Thus, M1–M3 and M5 were unambiguously identified as ginsenosides Rd, G-XVII, F2, and Cpd K, respectively. M4 was eluted at 39.7 min. The MS spectral profile of M4 was similar to those of M3 (ginsenoside F2) and ginsenoside Rg3 (Table 1), supporting that M4 contained a ginsenoside glycoside with two glucoses. However, the retention time of M4 was different from that of either ginsenoside Rg3 (40.3 min, with two glucoses at the C-3 position) or F2 (38.2 min, with one glucose at the C-3 position and another at the C-20 position) (Figure 1). Thus, M4 was tentatively identified as G-LXXV, a metabolite formed from Rb1 with two glucose molecules remaining at the C-20 position. Taken together, Rb1 biotransformation by human gut bacteria in vitro resulted in the formations of gypenosides as well as metabolites of the Rd pathway. Time-course of Rb1 metabolism by human gut bacteria
The calibration curves of Rb1, Rd, F2, G-XVII, and Cpd K in the in vitro anaerobic incubation system showed good linearity (r2 > 0.998, P < 0.001) over the concentration ranges tested (Table 2). The overall intra-day and inter-day variations were less than 6%, and recovery was higher than 85% (except for ~60% for 10 μM Cpd K) (Additional file 1: Table S1). When Rb1 was incubated with the pooled human gut bacteria (Figure 2A), Rb1 was rapidly eliminated by the human gut bacteria and less than 20% of the Rb1 remained intact at 12 h. The metabolites from the Rd pathway, Rd and F2, appeared in the incubations in turn, with Rd reaching its maximum at 12 h (representing ~50% of the initial
Table 1 HPLC-ESI-MS and corresponding CID data of ginsenoside standards and the metabolites of ginsenoside Rb1 by human gut bacteria Retention time (min)
MS (m/z)
CID (m/z)
Identity
859
637[M-H-Glc]-, 619[M-H-Glc-H2O]-, 475[M-H-2Glc]-
Ginsenoside Rg1 (IS)
1143
-
945[M-H-Glc]-, 783[M-H-2Glc]-, 621[M-H-3Glc]-
Ginsenoside Rb1
981
-
783[M-H-Glc]-, 621[M-H-2Glc]-,
Ginsenoside Rd (M1)
981
-
783[M-H-Glc]-, 621[M-H-2Glc]-,
Gypenoside XVII (M2)
819
843
621[M-H-Glc]-, 459[M-H-2Glc]-
Ginsenoside F2 (M3)
[M-H]-
[M + Cl]-
[M + AcO]-
20.9
799
835
33.2
1107
35.1
945
35.6
945
38.2
783
-
-
39.7
783
819
843
621[M-H-Glc] , 459[M-H-2Glc]
40.3
783
819
-
621[M-H-Glc]-, 459[M-H-2Glc]-
45.4
621
657
681
459[M-H-Glc]-
Ginsenoside Rh2
44.4
621
657
681
459[M-H-Glc]-
Compound K (M5)
Gypenoside LXXV (M4) Ginsenoside Rg3
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
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Table 2 Calibration curves of ginsenosides in simulated gastric fluid and an in vitro human gut bacteria incubation system Matrix Simulated gastric fluid
Human gut bacteria
Analyte
Calibration curve (μΜ)
r2
Rb1
Y = 3.504X + 6.912
0.9999
2.08-167.00
Rd
Y = 3.796X-3.843
0.9999
11.30-3618.00
Concentration range (μΜ)
G-XVII
Y = 3.549X-3.430
0.9999
5.21-1666.67
F2
Y = 3.339X-1.033
0.9999
5.21-1666.67
Rg3
Y = 3.377X + 10.180
0.9998
1.85-148.30
Cpd K
Y = 3.807X + 0.303
0.9999
5.21-1666.67
Rh2
Y = 3.644X + 6.880
0.9999
5.21-1666.67
Rb1
Y = 6.1209X + 0.094
0.9986
50.00-800.00
Rd
Y = 7.3891X + 4E-0.5
0.9998
31.25-500.00
G XVII
Y = 6.1571X + 0.062
0.9999
30.00-200.00
F2
Y = 6.7242X + 0.003
0.9999
16.25-260.00
Cpd K
Y = 6.7841X + 0.042
0.9945
5.16-165.00
Rb1) followed by the maximum of F2 at relatively low levels (representing ~7% of the initial Rb1). The amount of Cpd K peaked at 36 h. Compared with their respective positional isomers Rd and F2, G-XVII and G-LXXV formed from the gypenoside pathway reached their maximum levels more rapidly (Tmax : 4 and 8-12 h, respectively). Because the positional isomers Rd and GXVII showed similar UV responses, as evidenced by the very close constant coefficients (Table 2), we assumed that F2 and G-LXXV, the positional isomers formed by
hydrolytic removal of two glucoses from C-3 and C-20 (F2) and C-3 (G-LXXV), would also have similar UV responses. Thus, the levels of the positional isomers F2 and G-LXXV were calculated using the calibration curve of F2. Both gypenosides were formed at relatively lower levels than their positional isomers. The maximum level of G-XVII was about 10% of Rd, while that of G-LXXV was half of the peak concentration of F2. Moreover, both gypenosides had disappeared from the incubation system within 24 h, long before Cpd K
Figure 2 Time-courses of the elimination and metabolite formations of (A) ginsenoside Rb1, and (B) Rd (solid line) and G-XVII (dotted line) in human gut bacteria.
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
reached its maximum level at 36 h, while Rd and F2 were still detectable at 48 h. Thus, the gypenoside pathway should be the minor pathway of Rb1 biotransformation by human gut bacteria. Biotransformation of Rd and G-XVII by human gut bacteria
Rd and G-XVII were depleted within 8 h by human gut bacteria, and generated F2 and Cpd K when incubated separately. The generations of F2 and Cpd K from Rd peaked at 2 and 12 h, respectively, while those generated from G-XVII reached their maximum levels at 4 and 18 h, respectively. The maximum levels of F2 and Cpd K formed from Rd were comparable to those from G-XVII (about 30 and 80 μM, reflecting 30% and 80% of the original amounts of the respective parent compounds) (Figure 2B).
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G-LXXV was also generated by human gut bacteria as evidenced by the retention time and mass spectral profile. F2 presented as a major metabolite and the Cmax was two times higher that of G-LXXV (Figure 2B). Therefore, both the G-XVII → F2 and G-XVII → G-LXXV pathways of G-XVII metabolism existed in the human gut bacteria, with the former as the major pathway in vitro. Rb1 biotransformation by individual human gut bacterial samples
When Rb1 was separately incubated with gut bacterial samples from 58 individuals aged 18-92 years (30 females and 28 males; range, 39.6-75.3 years), great inter-individual variations were observed in terms of the remaining Rb1 and the amounts of metabolites formed via the Rd and gypenoside pathways (Figure 3A). However, when compared by age, there were no significant differences in Rb1
Figure 3 Ginsenoside Rb1 metabolism and its metabolite formation by gut bacteria samples from 58 healthy individuals (A) of different ages (B) and sexes (C).
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elimination and each pathway among individuals aged 18-45 years (20 individuals; range, 29.2-47 years), 4670 years (22 individuals; range, 52.7-65.9 years), and 7192 years (16 individuals; range, 72.6-85.4 years) (Figure 3B). No sex differences (P values: 0.3 ~ 0.5) were observed in Rb1 metabolism and each pathway by human gut bacteria (Figure 3C).
Degradation of Rb1 and Rd in SGF
The calibration curves of Rb1, Rd, G-XVII, F2, Rg3, Cpd K, and Rh2 in SGF showed good linearity (r2 > 0.998, P < 0.001) over the concentration ranges tested (Table 2). Rb1 was rapidly degraded under acidic environments to half of its original amount in 1 h, with
RESEARCH
Open Access
Biotransformation of ginsenoside Rb1 via the gypenoside pathway by human gut bacteria Hong Shen1,2†, Weng-Im Leung1†, Jian-Qing Ruan1, Song-Lin Li2, Jacky Pui-Cheong Lei3, Yi-Tao Wang1 and Ru Yan1*
Abstract Background: Bacterial conversion of ginsenosides is crucial for the health-promoting effects of ginsenosides. Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria in vitro. Methods: The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis. Results: When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process. Conclusions: Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria in vitro.
Background Ginsenosides are the major bioactive components in Panax, including Panax ginseng, Panax quinquefolium, and Panax notoginseng [1], which are the most popular herbs used for medicinal and nutritional purposes in China, Japan, Korea, and some Western countries. Two dammarane-type triterpene O-glycosides, namely 20(S)protopanaxadiol (PPD) and 20(S)-protopanaxatriol, are the major ginsenosides in different parts of Panax, including the flower bud, berry, crown, rootlet, side root, seed bud, seed, stem, and leaf [2,3], and their differently processed herbal products [4]. As one of the most important PPDtype ginsenosides, ginsenoside Rb1 (Rb1) has exhibited * Correspondence: [email protected] † Equal contributors 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China Full list of author information is available at the end of the article
various pharmacological activities, including neuroprotective, antitumor, cardiovascular-protective, and anti-aging effects, in many in vitro and in vivo models [5-7]. The intestinal absorption of glycosides in plants or foods is poor, while their metabolites generated by intestinal microflora are more permeable and bioactive [8-11]. The oral bioavailability of PPD-type ginsenosides is generally low (98% (HPLCUV). The BBL™ Brain Heart Infusion (BHI) medium,
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GasPak™ EZ anaerobe container system with an indicator, and GasPak™ EZ large incubation container were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). L-cystine was purchased from Research Organics Inc. (Cleveland, OH, USA). Dimethyl sulfoxide (DMSO), bovine hemin, and vitamin K1 were supplied by SigmaAldrich (St. Louis, MO, USA). Methanol, 1-butanol, and acetonitrile were HPLC-grade and purchased from Merck (Darmstadt, Germany). Dulbecco’s phosphate-buffered saline (PBS) was provided by Life Technologies (Carlsbad, CA, USA). Hydrochloric acid and sodium carbonate (analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared in-house using a Milli-Q purification system (Millipore, Bedford, MA, USA). Instrumentation and analytical conditions
Chromatographic separation was performed on an Agilent 1200 Series HPLC apparatus (Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, a binary pump, and an autosampler, and connected to a diode array detector (DAD). Samples were loaded onto an Alltima C18 column (250 × 4.6 mm, 5 μm). The column temperature was maintained at 25°C. The mobile phase consisted of water (A) and acetonitrile (B) and flowed at 1.5 mL/min. The following gradient elution program was used for samples obtained from bacterial incubations: 010 min, 19-20% B; 10-28 min, 20-30% B; 28-40 min, 3060% B; 40-48 min, 60-100% B. The elution program was modified when the incubations of ginsenosides with SGF were analyzed as follows: 0-10 min, 19-20% B; 10-28 min, 20-30% B; 28-35 min, 30-60% B; 35-45 min, 60-100% B. Rb1 and its bacterial metabolites were monitored at 203.4 nm and their UV absorbance was recorded over 200-400 nm. The injection volume was 5 μL. MS analysis was performed on an liquid chromatograph/mass selective detector (LC/MSD) Trap system (Agilent Technologies, Palo Alto, CA, USA) equipped with an ion-trap mass spectrometer with an electrospray ionization (ESI) interface. Agilent ChemStation for LC 3D System software (Rev. B. 01. 03-SR2) (Agilent Technologies, Santa Clara, CA, USA) was used for instrument control, data acquisition, and data management. Except for replacement of the water with 50 mM ammonium acetate in water, the mobile phase composition and gradient elution program were the same as those for the above HPLC analysis method for human gut bacteria samples. The mass spectrometer was operated in the negative ion mode with the following conditions: drying gas (N2), 8 L/min; temperature, 325°C; nebulizer pressure, 30 psi; scan range, m/z 100-1400. The ESIMS/MS conditions were as follows: negative ion mode; separation width, 4; fragment amplification, 1.0 V; scan range, m/z 100-1400.
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
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Preparation of human gut bacteria
Inter-individual variations in the gypenoside and Rd pathways were investigated by incubating Rb1 with gut bacteria suspensions from different individuals under the same conditions described above, and the incubated samples collected at 18 h were processed as described above. Samples were analyzed by HPLC-UV, and the peak area ratio data of each analyte were collected for comparison.
The medium was prepared according to our previous report [33]. Briefly, 100 mL of autoclaved BHI medium (3.7 g/100 mL) was supplemented with 0.05 mg of vitamin K1, 0.5 mg of bovine hemin, and 50 mg of L-cystine. The fecal samples (healthy Chinese, 18-92 years old) were provided by Kiang Wu Hospital and the experimental protocol is approved by Kiang Wu Hospital and University of Macau before conduct. Human gut bacteria suspensions were prepared at 4°C according to our previous report [33] with minor modifications. Fecal samples were freshly collected and pooled at 1 g each for identification of Rb1 metabolic pathways, or individually processed at 5 g each for characterization of individual variations in Rb1 metabolism. The individual or pooled fecal samples were mixed with 20 mL of BHI medium and the resulting suspensions were centrifuged at 200 × g for 5 min. The supernatants were then centrifuged at 5000 × g for 30 min. The human gut bacteria suspensions were obtained by resuspension of the bacterial precipitates in 5 mL of BHI medium. Biotransformation of Rb1 by human gut bacteria
Biotransformation assays of Rb1 were conducted with pooled or individual human gut bacteria samples to examine the existence of the gypenoside pathway and compare it with the Rd pathway. A high concentration of Rb1 was used to capture the minor formation and quantitative variations in the metabolites formed from the minor gypenoside pathway based on the great individual variations in gut bacteria compositions and metabolic capabilities [34]. The anaerobic incubation system contained 250 μL of gut bacteria suspension, 100 μL of Rb1 in DMSO (final concentration, 100 mM Rb1), and 4.75 mL of BHI medium. The incubation system was maintained in an anaerobic state at 37°C using the GasPak™ EZ anaerobe container system. The metabolic pathway and time-course of Rb1 metabolism by human gut bacteria were characterized in five independent experiments using pooled samples. In each experiment, the reaction of Rb1 with a human gut bacteria suspension was conducted in duplicate. Control reactions were conducted in parallel by adding 250 μL of PBS (pH 7.0) instead of gut bacteria suspension. The reactions were terminated at 0, 2, 4, 12, 18, 24, 36, and 48 h, respectively, by adding 15 mL of 1-butanol followed by immediate centrifugation at 5000 × g for 30 min to remove the bacteria. The resulting supernatants were mixed with 100 μL of ginsenoside Rg1 (final concentration, 100 mM) as an internal standard (IS), and each mixture was extracted twice with water-saturated 1-butanol. The organic layers were combined and dried by rotary evaporation (BUCHI, Flavil, Switzerland). The residue was reconstituted in 0.2 mL of methanol, and an aliquot (5 μL) was subjected to HPLC-UV or HPLC-MS analysis.
Biotransformation of Rd and G-XVII by human gut bacteria
The metabolism of the two positional isomers Rd and G-XVII by human gut bacteria was investigated by incubation with the same human gut bacteria sample in parallel to identify the pathways between Rd/G-XVII and Cpd K. The anaerobic incubation system contained 20 μL of gut bacteria suspension, 10 μL of ginsenoside Rd or G-XVII (final concentration, 100 μM Rd or GXVII), and 170 μL of BHI medium. The concentrations of Rd and G-XVII were determined based on the high amounts of all metabolites determined from a preliminary study. Each reaction was conducted in triplicate. Control reactions without gut bacteria were conducted in parallel. Reactions were terminated at 0, 2, 4, 6, 8, 12, 18, 24, 36, and 48 h, respectively, by adding 1.2 mL of 1-butanol followed by centrifugation at 5000 × g for 30 min to remove the bacteria. After adding 10 μL of ginsenoside Rg1 (final concentration, 100 μM Rg1) as an internal standard, the resulting mixture was processed in the same manner described above before being subjected to HPLC-UV analysis. Stabilities of Rb1 and Rd in SGF
SGF was prepared by diluting 3.84 mL of hydrochloric acid to 1000 mL with deionized water (pH 1.2) according to the Chinese Pharmacopoeia [35]. Ten microliters of ginsenoside Rb1 or Rd in DMSO (final concentration, 71.4 μM) was mixed with 200 μL of SGF, and kept at 37°C for different time intervals (0, 20, 40, 60, 90, 150, 180 and 300 min) before adding 70 μL of 0.1 mol/L Na2CO3 to examine the acidic stability. Each reaction was conducted in triplicate. The mixture was shaken for 1 min by vortexing and centrifuged at 14,000 × g for 5 min. The upper layer was decanted and an aliquot (20 μL) was subjected to HPLC-UV analysis. Calibration curves of Rb1 and its metabolites Rd, F2, G-XVII, and Cpd K in human gut bacteria
Serial concentrations of Rb1, Rd, G-XVII, F2, and Cpd K were prepared in DMSO, and mixed with a pooled human gut bacteria suspension and BHI medium as described in the section entitled Biotransformation of Rb1 by Human Gut Bacteria. The resulting mixtures were
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
immediately extracted with 1-butanol and centrifuged (5000 × g, 30 min). The organic layers were processed in the same manner described above and then subjected to HPLC-UV analysis. Calibration curves were constructed by plotting the peak area ratio of each analyte to the IS (Y) as a function of the analyte concentration (X) in the reaction system. For measurements of the intra-day precision and accuracy, three concentration levels for each standard (low, medium, and high) were prepared as quality control (QC) samples and analyzed on the same day. The inter-day
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precision and accuracy were determined with the same three QC samples on three consecutive days. Calibration curves of ginsenosides in SGF
A mixed stock solution of Rb1, Rd, G-XVII, F2, Rg3, Cpd K, and Rh2 was prepared in DMSO. A series of working solutions were prepared with DMSO using dilution factors of 2.5, 5, 10, 20, 40, and 80 from the stock solution. An aliquot (10 μL) of each working solution was spiked with 200 μL of SGF and processed with 70 μL of 0.1 mol/L Na2CO3 as described above. The
Figure 1 HPLC-UV chromatograms of (A) mixed ginsenoside standards, and incubated samples of Rb1 with (B) pooled gut bacteria and intestinal bacteria from (C) a 65-year-old male, (D) a 44-year-old male, and (E) a 54-year-old female. M1, ginsenoside Rd; M2, gypenoside XVII; M3, ginsenoside F2; M4, gypenoside LXXV; M5, compound K.
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sample (20 μL) was injected into the HPLC instrument. A calibration curve was obtained by plotting the peak area of each analyte (Y) against its concentration in SGF (X). Data analysis
Data were presented as the means ± standard deviation (SD) of triplicate analyses (experiments with pooled human gut bacteria or SGF) or individual measurements (experiments on inter-individual variations). Sex and age differences in ginsenoside Rb1 metabolism were compared by Student’s t-test. Values of P < 0.05 were considered statistically significant.
Results Metabolism of ginsenoside Rb1 by pooled human gut bacteria
Fecal samples from 50 healthy volunteers (25 males and 25 females, range, 45.7-73.7 years) were collected and pooled at 10 individuals each to prepare five human gut bacteria pools. When Rb1 was incubated with pooled human gut bacteria, five peaks (M1-M5) were observed at 35.1, 35.6, 38.2, 39.7, and 44.4 min, respectively, which were absent in control reactions (Figure 1). Under the developed analytical conditions, Rb1 and its metabolites were well-separated from the endogenous interferences of the in vitro anaerobic incubation system. The retention times and characteristic CID fragment ions of the ginsenoside standards and Rb1 metabolites are summarized in Table 1. Rb1 was eluted at 33.2 min and showed its pseudomolecular ion at m/z 1107 ([M-H]-) and chloride adduct ion at m/z 1143 ([M + Cl]-), corresponding to a molecular weight of 1108 Daltons. The fragment ions at m/z 945 ([M-H2O-H]-), m/z 783 ([M-2H2O-H]-), and m/z 621 ([M-3H2O-H]-) in the CID spectrum corresponded to loss of one, two, and three glucose molecules from Rb1, respectively.
The retention times and mass spectral profiles of M1 (35.1 min), M3 (38.2 min), and M5 (44.4 min) were identical to those of Rd, F2, and Cpd K, respectively. The retention time and mass spectral profile of M2 (35.6 min) were identical to those of G-XVII, which was formed from Rb1 by hydrolytic removal of one glucose molecule from the C-3 position of Rb1. Thus, M1–M3 and M5 were unambiguously identified as ginsenosides Rd, G-XVII, F2, and Cpd K, respectively. M4 was eluted at 39.7 min. The MS spectral profile of M4 was similar to those of M3 (ginsenoside F2) and ginsenoside Rg3 (Table 1), supporting that M4 contained a ginsenoside glycoside with two glucoses. However, the retention time of M4 was different from that of either ginsenoside Rg3 (40.3 min, with two glucoses at the C-3 position) or F2 (38.2 min, with one glucose at the C-3 position and another at the C-20 position) (Figure 1). Thus, M4 was tentatively identified as G-LXXV, a metabolite formed from Rb1 with two glucose molecules remaining at the C-20 position. Taken together, Rb1 biotransformation by human gut bacteria in vitro resulted in the formations of gypenosides as well as metabolites of the Rd pathway. Time-course of Rb1 metabolism by human gut bacteria
The calibration curves of Rb1, Rd, F2, G-XVII, and Cpd K in the in vitro anaerobic incubation system showed good linearity (r2 > 0.998, P < 0.001) over the concentration ranges tested (Table 2). The overall intra-day and inter-day variations were less than 6%, and recovery was higher than 85% (except for ~60% for 10 μM Cpd K) (Additional file 1: Table S1). When Rb1 was incubated with the pooled human gut bacteria (Figure 2A), Rb1 was rapidly eliminated by the human gut bacteria and less than 20% of the Rb1 remained intact at 12 h. The metabolites from the Rd pathway, Rd and F2, appeared in the incubations in turn, with Rd reaching its maximum at 12 h (representing ~50% of the initial
Table 1 HPLC-ESI-MS and corresponding CID data of ginsenoside standards and the metabolites of ginsenoside Rb1 by human gut bacteria Retention time (min)
MS (m/z)
CID (m/z)
Identity
859
637[M-H-Glc]-, 619[M-H-Glc-H2O]-, 475[M-H-2Glc]-
Ginsenoside Rg1 (IS)
1143
-
945[M-H-Glc]-, 783[M-H-2Glc]-, 621[M-H-3Glc]-
Ginsenoside Rb1
981
-
783[M-H-Glc]-, 621[M-H-2Glc]-,
Ginsenoside Rd (M1)
981
-
783[M-H-Glc]-, 621[M-H-2Glc]-,
Gypenoside XVII (M2)
819
843
621[M-H-Glc]-, 459[M-H-2Glc]-
Ginsenoside F2 (M3)
[M-H]-
[M + Cl]-
[M + AcO]-
20.9
799
835
33.2
1107
35.1
945
35.6
945
38.2
783
-
-
39.7
783
819
843
621[M-H-Glc] , 459[M-H-2Glc]
40.3
783
819
-
621[M-H-Glc]-, 459[M-H-2Glc]-
45.4
621
657
681
459[M-H-Glc]-
Ginsenoside Rh2
44.4
621
657
681
459[M-H-Glc]-
Compound K (M5)
Gypenoside LXXV (M4) Ginsenoside Rg3
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Table 2 Calibration curves of ginsenosides in simulated gastric fluid and an in vitro human gut bacteria incubation system Matrix Simulated gastric fluid
Human gut bacteria
Analyte
Calibration curve (μΜ)
r2
Rb1
Y = 3.504X + 6.912
0.9999
2.08-167.00
Rd
Y = 3.796X-3.843
0.9999
11.30-3618.00
Concentration range (μΜ)
G-XVII
Y = 3.549X-3.430
0.9999
5.21-1666.67
F2
Y = 3.339X-1.033
0.9999
5.21-1666.67
Rg3
Y = 3.377X + 10.180
0.9998
1.85-148.30
Cpd K
Y = 3.807X + 0.303
0.9999
5.21-1666.67
Rh2
Y = 3.644X + 6.880
0.9999
5.21-1666.67
Rb1
Y = 6.1209X + 0.094
0.9986
50.00-800.00
Rd
Y = 7.3891X + 4E-0.5
0.9998
31.25-500.00
G XVII
Y = 6.1571X + 0.062
0.9999
30.00-200.00
F2
Y = 6.7242X + 0.003
0.9999
16.25-260.00
Cpd K
Y = 6.7841X + 0.042
0.9945
5.16-165.00
Rb1) followed by the maximum of F2 at relatively low levels (representing ~7% of the initial Rb1). The amount of Cpd K peaked at 36 h. Compared with their respective positional isomers Rd and F2, G-XVII and G-LXXV formed from the gypenoside pathway reached their maximum levels more rapidly (Tmax : 4 and 8-12 h, respectively). Because the positional isomers Rd and GXVII showed similar UV responses, as evidenced by the very close constant coefficients (Table 2), we assumed that F2 and G-LXXV, the positional isomers formed by
hydrolytic removal of two glucoses from C-3 and C-20 (F2) and C-3 (G-LXXV), would also have similar UV responses. Thus, the levels of the positional isomers F2 and G-LXXV were calculated using the calibration curve of F2. Both gypenosides were formed at relatively lower levels than their positional isomers. The maximum level of G-XVII was about 10% of Rd, while that of G-LXXV was half of the peak concentration of F2. Moreover, both gypenosides had disappeared from the incubation system within 24 h, long before Cpd K
Figure 2 Time-courses of the elimination and metabolite formations of (A) ginsenoside Rb1, and (B) Rd (solid line) and G-XVII (dotted line) in human gut bacteria.
Shen et al. Chinese Medicine 2013, 8:22 http://www.cmjournal.org/content/8/1/22
reached its maximum level at 36 h, while Rd and F2 were still detectable at 48 h. Thus, the gypenoside pathway should be the minor pathway of Rb1 biotransformation by human gut bacteria. Biotransformation of Rd and G-XVII by human gut bacteria
Rd and G-XVII were depleted within 8 h by human gut bacteria, and generated F2 and Cpd K when incubated separately. The generations of F2 and Cpd K from Rd peaked at 2 and 12 h, respectively, while those generated from G-XVII reached their maximum levels at 4 and 18 h, respectively. The maximum levels of F2 and Cpd K formed from Rd were comparable to those from G-XVII (about 30 and 80 μM, reflecting 30% and 80% of the original amounts of the respective parent compounds) (Figure 2B).
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G-LXXV was also generated by human gut bacteria as evidenced by the retention time and mass spectral profile. F2 presented as a major metabolite and the Cmax was two times higher that of G-LXXV (Figure 2B). Therefore, both the G-XVII → F2 and G-XVII → G-LXXV pathways of G-XVII metabolism existed in the human gut bacteria, with the former as the major pathway in vitro. Rb1 biotransformation by individual human gut bacterial samples
When Rb1 was separately incubated with gut bacterial samples from 58 individuals aged 18-92 years (30 females and 28 males; range, 39.6-75.3 years), great inter-individual variations were observed in terms of the remaining Rb1 and the amounts of metabolites formed via the Rd and gypenoside pathways (Figure 3A). However, when compared by age, there were no significant differences in Rb1
Figure 3 Ginsenoside Rb1 metabolism and its metabolite formation by gut bacteria samples from 58 healthy individuals (A) of different ages (B) and sexes (C).
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elimination and each pathway among individuals aged 18-45 years (20 individuals; range, 29.2-47 years), 4670 years (22 individuals; range, 52.7-65.9 years), and 7192 years (16 individuals; range, 72.6-85.4 years) (Figure 3B). No sex differences (P values: 0.3 ~ 0.5) were observed in Rb1 metabolism and each pathway by human gut bacteria (Figure 3C).
Degradation of Rb1 and Rd in SGF
The calibration curves of Rb1, Rd, G-XVII, F2, Rg3, Cpd K, and Rh2 in SGF showed good linearity (r2 > 0.998, P < 0.001) over the concentration ranges tested (Table 2). Rb1 was rapidly degraded under acidic environments to half of its original amount in 1 h, with
